RNA splicing, studied here using the group II intron as a model system, is a central component of gene expression and malfunctions in this process can have disasterous effects on protein expression. Many human diseases are caused by mutations that interfere with RNA splicing. To understand the basis of these effects, more information is required with regard to the fundamental chemistry of splicing. Moreover, it is essential to elucidate the three-dimensional structure of the group II intron since its structure is critical for its function. To this end, UV and photoaffinity crosslinking, affinity cleavage and modification footprinting experiments will be used to generate distance constraints. These distance constraints, along with secondary structure data will be used in a molecular modeling protocol to develop a three-dimensional model of the active sites for the hydrolysis and transesterification reactions promoted by the group II intron. Because unusual tertiary interactions appear to be solely responsible for folding and organizing the active site of the group II intron its structure cannot be understood using phylogenetic covariation or genetic approaches. Ribozymes are also potentially promising as potential therapeutic agents because they can specifically target and cleave undesirable RNA gene products. One approach to human gene therapy involves the introduction of DNA coding for a ribozyme into an organism, where it is transcribed and then goes on to cleave specific undesirable RNAs in the cell. However, much needs to be learned about the reactivity and structure of ribozymes before they can be successfully applied in gene therapy. Since group II introns are both ribozymes and simple models of pre-mRNA splicing in eukaryotes, an understanding of their structure and function may lead to advances in medical research at many different levels.